Polynucleotide encoding saliva binding protein

ABSTRACT

Saliva binding protein polypeptides and DNA (RNA) of Staphylococcus aureus encoding such saliva binding protein and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such saliva binding protein for the treatment of infection, particularly bacterial infections. Antagonists against such saliva binding protein and their use as a therapeutic to treat infections, particularly bacterial infections are also disclosed. Also disclosed are diagnostics assays for detecting diseases related to the presence of saliva binding protein nucleic acid sequences and the polypeptides in a host. Also disclosed are diagnostic assays for detecting polynucleotides encoding saliva binding protein family and for detecting the polypeptide in a host.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides and recombinant host cells transformed with thepolynucleotides. The invention also relates to inhibiting the action ofsuch polypeptides and to the use of inhibitors in therapy.

BACKGROUND OF THE INVENTION

Several cell surface associated proteins of the Staphylococci andStreptococci involved in microbial adhesion to different host tissuesand considered to be important factors in bacterial pathogenesis havebeen identified in the last decade (see Patti, J. M., Allen, B. L.,McGavin, M. J. and Hook, M., MSCRAMM-Mediated Adherence ofMicroorganisms to Host Tissues 1994! Annu.Rev.Microbiol. 48,585-617.).

The cell surface promin Saliva Binding Protein of Streptococcus sanguis(Ganeshkumar et al., 1991! Infect. Immun. 59, 1093-1099) is thought tobe an adhesin which plays a role in oral colonisation by Streptococcussanguis.

Different approaches have been put forward to address such proteins fromStaphylococcus aureus as antibacterial targets, e.g. fibronectin bindingproteins (EP0294349, EP0397633, WO94/18327), fibrinogen binding protein(WO94/06830), collagen binding promin (WO92/07002) and bone sialoproteinbinding protein (WO94/13310). The binding proteins or binding fragmentsthereof are used as antibacterial agents to block binding of theorganism to host tissue, as vaccines to raise antibodies to the organismin the host animal or as antigens to raise therapeutic antibodies whichcan be used to block binding of the organism to host tissue.

Recently several novel approaches have been described which purport tofollow global gene expression during infection (Chung, S. et al. 1993!Global Regulation of Gene Expression in Escherichia coli J. Bacteriol.175, 2026-2036, Mahan, M. J. et al. 1993! Selection of BacterialVirulence Genes That Are Specifically Induced in Host Tissues SCIENCE259, 686-688, Hensel, M. et al. 1995! Simultaneous Identification ofBacterial Virulence Genes by Negative Selection SCIENCE 269, 400-403).These new techniques have so far been demonstrated with gram negativepathogen infections and not with infections with gram positivespresumably due to the much slower development of global transposonmutagenesis and suitable vectors needed for these strategies in theseorganisms, and in the case of that process described by Chuang, S. etal. 1993! the difficulty of isolating suitable quantities of bacterialRNA free of mammalian RNA derived from the infected tissue to furnishbacterial RNA labelled to sufficiently high specific activity. Thepresent invention employs a novel technology to determine geneexpression in the pathogen at different stages of infection of themammalian host. A novel aspect of this invention is the use of asuitably labelled oligonucleotide probe which anneals specifically tothe bacterial ribosomal RNA in Northern blots of bacterial RNApreparations from infected tissue. Using the more abundant ribosomal RNAas a hybridisation target greatly facilitates the optimisation of aprotocol to purify bacterial RNA of a suitable size and quantity forRT-PCR from infected tissue.

A suitable oligonucleotide useful for applying this method to genesexpressed in Staphylococcus aureus is

    5'-gctcctaaaaggttactccaccggc-3'

Use of the technology of the present invention enables identification ofbacterial genes transcribed during infection, inhibitors of which wouldhave utility in anti-bacterial therapy. Specific inhibitors of such genetranscription or of the subsequent translation of the resultant mRNA orof the function of the corresponding expressed proteins would haveutility in anti-bacterial therapy.

SUMMARY OF THE INVENTION

The present invention relates to a novel cell surface protein from S.aureus WCUH 29, characterised in that it comprises the amino acidsequence given in SEQ ID NO 1, or a fragment, analogue or derivativethereof.

The invention also relates to a polypeptide fragment of the cell surfaceprotein, having the amino acid sequence given in SEQ ID NO 1, or aderivative thereof.

In accordance with another aspect of the present invention, there areprovided, polynucleotides (DNA or RNA) which encode such polypeptides.

In particular the invention provides a polynucleotide having the DNAsequence given in SEQ ID NO 2.

The present invention also provides a novel protein from Staphylococcus.aureus WCUH29 obtainable by expression of a gene characterised in thatit comprises the DNA sequence given SEQ ID NO 2, or a fragment, analogueor derivative thereof.

The invention also relates to novel oligonucleotides, including SEQ IDNOs 3 and 4, derived from the sequences SEQ ID NO 2.

The present invention includes variants of the hereinabove describedpolynucleotides which encode fragments, analogs and derivatives of thepolypeptide characterised by the deduced amino acid sequence of SEQ IDNO 1.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Also provided is an antibody against the polypeptide of SEQ ID NO: 1.Still further provided is an antagonist which inhibits the activity ofthe polypeptide of SEQ ID NO: 1.

A method is also provided for the treatment of an individual having needto inhibit novel saliva binding protein polypeptide comprising:administering to the individual a therapeutically effective amount of anantagonist against the polypeptide of the invention.

Provided is a process for diagnosing a disease related to expression ofthe polypeptide of the invention comprising: determining a nucleic acidsequence encoding the polypeptide of SEQ ID NO: 1.

A diagnostic process is provided comprising: analyzing for the presenceof the polypeptide of SEQ ID NO: 1 in a sample derived from a host.

In accordance with yet a further aspect of the present invention, thereis provided the use of a polypeptide of the invention for therapeutic orprophylactic purposes, for example, as an antibacterial agent or avaccine.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunisation.

In accordance with yet another aspect of the present invention, thereare provided inhibitors to such polypeptides, useful as antibacterialagents.

Another aspect of the invention is a pharmaceutical compositioncomprising the above polypeptide, polynucleotide or inhibitor of theinvention and a pharmaceutically acceptable carrier.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the immediate physical interaction between a pathogen and mammalianhost responsible for sequelae of infection.

The invention further relates to the manufacture of a medicament forsuch uses.

This invention provides a method of screening drugs to identify thosewhich interfere with the interaction of the cell surface protein oractive fragment to mammalian cells.

Further provided is a method for identifying compounds which bind to andinhibit an activity of the polypeptide of SEQ ID NO: 1 comprising:contacting a cell expressing on the surface thereof a binding means forthe polypeptide, said binding means being associated with a secondcomponent capable of providing a detectable signal in response to thebinding of a compound to said binding means, with a compound to bescreened under conditions to permit binding to the binding means; anddetermining whether the compound binds to and activates or inhibits thebinding by detecting the presence or absence of a signal generated fromthe interaction of the compound with the binding means.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the polypeptide sequence of novel saliva binding proteinSEQ ID NO:1!.

FIGS. 2, 2a and 2b shows the polynucleotide sequence of novel Novelsaliva binding protein SEQ ID NO: 2! deduced from the polynucleotidesequence of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel cell surface protein from S.aureus WCUH 29, characterised in that it comprises the amino acidsequence given in SEQ ID NO 1, or a fragment, analogue or derivativethereof.

Staphylococcus aureus WCUH 29 has been deposited at the NationalCollection of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen,Scotland under number NCIMB 40771 on 11 Sep. 1995.

The invention also relates to a protein having the amino acid sequencegiven in SEQ ID NO 1, or a derivative thereof. The amino acid sequenceof SEQ ID NO 1 displays 47% identity over 309 amino acids to theSaliva-Binding Protein of Streptococcus sanguis (SSAB₋₋ STRPA) and withthe same level of homology to the Enterococcus faecalis endocarditisspecific antigen gene (EFU03756, Lowe et al., 1995! Infect. Immun. 63,703-706).

Hereinafter the term polypeptide(s) will be used to refer to the cellsurface protein and its fragments, analogues or derivatives.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In particular the invention provides a polynucleotide having the DNAsequence given in SEQ ID NO 2. The invention further provides apolynucleotide encoding a cell surface protein from S. aureus WCUH 29and characterised in that it comprises the DNA sequence given in SEQ IDNO 2.

The present invention also provides a novel protein from Staphylococcus.aureus WCUH29 obtainable by expression of a gene having the DNA sequencegiven SEQ ID NO 2, or a fragment, analogue or derivative thereof.

The invention also relates to novel oligonucleotides, including SEQ IDNOs 3 and 4, derived from the sequences SEQ ID NO 2 which can act as PCRprimers in the process herein described to determine whether or not theStaphylococcus aureus genes identified herein in whole or in part aretranscribed in infected tissue. It is recognised that such sequenceswill also have utility in diagnosis of the stage of infection and typeof infection the pathogen has attained.

The polynucleotide having the DNA sequence given in SEQ ID NO 2 wasobtained from the sequencing of a library of clones of chromosomal DNAof S. aureus WCUH 29 in E. coli. It has been demonstrated by the processherein described that it is transcribed in vivo in an establishedinfection of S. aureus WCUH29 in a mouse model of infection.

To obtain the polynucleotide encoding the cell surface protein using theDNA sequence given in SEQ ID NO 2 typically a library of clones ofchromosomal DNA of S. aureus WCUH 29 in E. coli or some other suitablehost is probed with a radiolabelled oligonucleotide, preferably a 17meror longer, derived from the sequence. Clones carrying DNA identical tothat of the probe can then be distinguished using high stringencywashes. By sequencing the individual clones thus identified withsequencing primers designed from the original sequence it is thenpossible to extend the sequence in both directions to determine the fullgene sequence. Conveniently such sequencing is performed using denatureddouble stranded DNA prepared from a plasmid clone. Suitable techniquesare described by Maniatis, T., Fritsch, E. F. and Sambrook, J. inMOLECULAR CLONING, A Laboratory Manual 2nd edition 1989 Cold SpringHarbor Laboratory. see Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70!.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the polypeptide may be identical to thecoding sequence shown in SEQ ID NO 2 or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same polypeptide.

The present invention includes variants of the hereinabove describedpolynucleotides which encode fragments, analogs and derivatives of thepolypeptide characterised by the deduced amino acid sequence of SEQ IDNO 1. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samepolypeptide characterised by the deduced amino acid sequence of SEQ IDNO 1 as well as variants of such polynucleotides which variants encodefor a fragment, derivative or analog of the polypeptide. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

The polynucleotide may have a coding sequence which is a naturallyoccurring allelic variant of the coding sequence characterised by theDNA sequence of SEQ ID NO 2. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The polynucleotide which encodes for the mature polypeptide, i.e. thenative cell surface protein, may include only the coding sequence forthe mature polypeptide or the coding sequence for the mature polypeptideand additional coding sequence such as a leader or secretory sequence ora proprotein sequence.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention therefore includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence). During post-translational modification of the peptide, amethionine residue at the NH₂ -terminus may be deleted. Accordingly,this invention contemplates the use of both the methionine-containingand the methionineless amino terminal variants of the protein of theinvention.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence at either the 5' or 3'terminus of the gene which allows for purification of the polypeptide ofthe present invention. The marker sequence may be a hexa-histidine tagsupplied by the pQE series of vectors (supplied commercially by QuiagenInc.) to provide for purification of the polypeptide fused to the markerin the case of a bacterial host.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably at least 70% identity between the sequences. The presentinvention particularly relates to polynucleotides which hybridize understringent conditions to the hereinabove-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which retain substantially the same biological function oractivity as the polypeptide characterised by the deduced amino acidsequence of SEQ ID NO 1. The invention also provides an isolatedpolynucleotide comprising a member selected from the group consistingof: a polynucleotide having at least a 70% identity to a polynucleotideencoding a polypeptide comprising amino acids of SEQ ID NO: 1; apolynucleotide which is complementary to the polynucleotide of (a); anda polynucleotide comprising at least 15 sequential bases of thepolynucleotide of (a) or (b).

The deposit referred to herein will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited material, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited material, and no suchlicense is hereby granted.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide characterised by the deduced amino acid sequence of SEQ IDNO 1, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide characterised bythe deduced amino acid sequence of SEQ ID NO 1 may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe polypeptide, such as a leader or secretory sequence or a sequencewhich is employed for purification of the polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

In accordance with yet a further aspect of the present invention, thereis therefore provided a process for producing the polypeptide of theinvention by recombinant techniques by expressing a polynucleotideencoding said polypeptide in a host and recovering the expressedproduct. Alternatively, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a cosmid, a phage, etc. Theengineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Suitable expression vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA. However, any other vector may be used as long asit is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in eukaryotic or prokaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as "control" elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. The polypeptides of the present inventioncan be expressed using, for example, the E. coli tac promoter or theprotein A gene (spa) promoter and signal sequence. Leader sequences canbe removed by the bacterial host in post-translational processing. See,e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397. Promoter regionscan be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers. Twoappropriate vectors are PKK232-8 and PCM7. Particular named bacterialpromoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the proteinsequences relative to the growth of the host cell. Regulatory sequencesare known to those of skill in the art, and examples include those whichcause the expression of a gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Other types of regulatory elements may also be present in thevector, for example, enhancer sequences.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the "control" of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the coding sequencesmay be desirable to achieve this end. For example, in some cases it maybe necessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; i.e., to maintainthe reading frame. The control sequences and other regulatory sequencesmay be ligated to the coding sequence prior to insertion into a vector,such as the cloning vectors described above. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably, a leader sequencecapable of directing secretion of translated protein into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. More particularly, the present invention alsoincludes recombinant constructs comprising one or more of the sequencesas broadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pET-3 vectors (Stratagene),pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pBlueBacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage l (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), a baculovirus insect cell system,, YCp19(Saccharomyces). See, generally, "DNA Cloning": Vols. I & II, Glover etal. ed. IRL Press Oxford (1985) (1987) and; T. Maniatis et al.("Molecular Cloning" Cold Spring Harbor Laboratory (1982).

In some cases, it may be desirable to add sequences which cause thesecretion of the polypeptide from the host organism, with subsequentcleavage of the secretory signal.

Polypeptides can be expressed in host cells under the control ofappropriate promoters. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y., (1989), the disclosure of which ishereby incorporated by reference.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Depending on the expression system and host selected, the polypeptide ofthe present invention may be produced by growing host cells transformedby an expression vector described above under conditions whereby thepolypeptide of interest is expressed. The polypeptide is then isolatedfrom the host cells and purified. If the expression system secretes thepolypeptide into growth media, the polypeptide can be purified directlyfrom the media. If the polypeptide is not secreted, it is isolated fromcell lysates or recovered from the cell membrane fraction. Where thepolypeptide is localized to the cell surface, whole cells or isolatedmembranes can be used as an assayable source of the desired geneproduct. Polypeptide expressed in bacterial hosts such as E. coli mayrequire isolation from inclusion bodies and refolding. Where the matureprotein has a very hydrophobic region (normally at the C-terminus) whichleads to an insoluble product of overexpression, it may be desirable toexpress a truncated protein in which the hydrophobic region has beendeleted. The selection of the appropriate growth conditions and recoverymethods are within the skill of the art.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A "double-stranded DNA molecule" refers to the polymeric form ofdeoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in adouble-stranded helix, both relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5' to 3' direction along thenontranscribed strand of DNA (i.e., the strand having the sequencehomologous to the mRNA).

A DNA "coding sequence of" or a "nucleotide sequence encoding" aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate regulatory sequences.

A "promoter sequence" is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bound at the 3' terminus by atranslation start codon (e.g., ATG) of a coding sequence and extendsupstream (5' direction) to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoterscontain Shine-Dalgarno sequences in addition to the -10 and -35consensus sequences.

DNA "control sequences" refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the expression (i.e., thetranscription and translation) of a coding sequence in a host cell.

A control sequence "directs the expression" of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

A "host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous DNA sequence.

A cell has been "transformed" by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eukaryotic cells, a stably transformed ortransfected cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenous DNA.

A "clone" is a population of cells derived from a single cell or commonancestor by mitosis. A "cell line" is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

A "heterologous" region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature.

In accordance with yet a further aspect of the present invention, thereis provided the use of a polypeptide of the invention for therapeutic orprophylactic purposes, for example, as an antibacterial agent or avaccine.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunisation.

The encoded protein upon expression can be used as a target for thescreening of antibacterial drugs, preferably for antagonists of novelsaliva binding protein. Additionally, the DNA sequences encoding theamino terminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

In accordance with yet another aspect of the present invention, thereare provided inhibitors to such polypeptides, useful as antibacterialagents. In particular, there are provided antibodies against suchpolypeptides.

Another aspect of the invention is a pharmaceutical compositioncomprising the above polypeptide, polynucleotide or inhibitor of theinvention and a pharmaceutically acceptable carrier.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the immediate physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used:

i) in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds;

ii) to block cell surface protein mediated mammalian cell invasion by,for example, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al. 1992! Infect. Immun. 60, 2211-7);

iii) to block bacterial adhesion between mammalian extracellular matrixproteins and bacterial cell surface proteins which mediate tissuedamage;

iv) to block the normal progression of pathogenesis in infectionsinitiated other than by the implantation of in-dwelling devices or byother surgical techniques.

The invention further relates to the manufacture of a medicament forsuch uses.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused e.g. by mechanical, chemical or thermal damage or by implantationof indwelling devices, or wounds in the mucous membranes, such as themouth, mammary glands, urethra or vagina.

The polypeptides or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The term antibodies also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

Antibodies generated against the polypeptides of the present inventioncan be obtained by direct injection of the polypeptides into an animalor by administering the polypeptides to an animal, preferably anonhuman. The antibody so obtained will then bind the polypeptidesitself. In this manner, even a sequence encoding only a fragment of thepolypeptides can be used to generate antibodies binding the whole nativepolypeptides. Such antibodies can then be used to isolate thepolypeptide from tissue expressing that polypeptide.

Polypeptide derivatives include antigenically or immunologicallyequivalent derivatives which form a particular aspect of this invention.

The term `antigenically equivalent derivative` as used hereinencompasses a polypeptide or its equivalent which will be specificallyrecognised by certain antibodies which, when raised to the protein orpolypeptide according to the present invention, interfere with theimmediate physical interaction between pathogen and mammalian host.

The term `immunologically equivalent derivative` as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

In particular derivatives which are slightly longer or slightly shorterthan the native cell surface protein or polypeptide fragment of thepresent invention may be used. In addition, polypeptides in which one ormore of the amino acid residues are modified may be used. Such peptidesmay, for example, be prepared by substitution, addition, orrearrangement of amino acids or by chemical modification thereof. Allsuch substitutions and modifications are generally well known to thoseskilled in the art of peptide chemistry.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

Using the procedure of Kohler and Milstein (1975 Nature 256,495-497),antibody-containing cells from the immunised mammal are fused withmyeloma cells to create hybridoma cells secreting monoclonal antibodies.

The hybridomas are screened to select a cell line with high bindingaffinity and favorable cross reaction with other staphylococcal speciesusing one or more of the original polypeptide and/or the fusion protein.The selected cell line is cultured to obtain the desired Mab.

Hybridoma cell lines secreting the monoclonal antibody are anotheraspect of this invention.

Alternatively phage display technology could be utilised to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

The antibody should be screened again for high affinity to thepolypeptide and/or fusion protein.

As mentioned above, a fragment of the final antibody may be prepared.

The antibody may be either intact antibody of M_(r) approx 150,000 or aderivative of it, for example a Fab fragment or a Fv fragment asdescribed in Skerra, A and Pluckthun, A (1988) Science 240 1038-1040. Iftwo antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The antibody of the invention may be prepared by conventional means forexample by established monoclonal antibody technology (Kohler, G. andMilstein, C. (1975), Nature, 256, 495-497) or using recombinant meanse.g. combinatorial libraries, for example as described in Huse, W. D. etal., (1989) Science 246, 1275-1281.

Preferably the antibody is prepared by expression of a DNA polymerencoding said antibody in an appropriate expression system such asdescribed above for the expression of polypeptides of the invention. Thechoice of vector for the expression system will be determined in part bythe host, which may be a prokaryotic cell, such as E. coli (preferablystrain B) or Streptomyces sp. or a eukaryotic cell, such as a mouseC127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous orunicellular fungi or insect cell. The host may also be a transgenicanimal or a transgenic plant for example as described in Hiatt, A etal., (1989) Nature 34, 76-78!. Suitable vectors include plasmids,bacteriophages, cosmids and recombinant viruses, derived from, forexample, baculoviruses and vaccinia.

The Fab fragment may also be prepared from its parent monoclonalantibody by enzyme treatment, for example using papain to cleave the Fabportion from the Fc portion.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the patient. For example, if the patient is humanthe antibody may most preferably be `humanised`; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The modification need not be restricted to one of `humanisation`; otherprimate sequences (for example Newman, R. et al. 1992, Biotechnology,10, 1455-1460) may also be used.

The humanised monoclonal antibody, or its fragment having bindingactivity, form a particular aspect of this invention.

This invention provides a method of screening drugs to identify thosewhich interfere with the interaction of the cell surface protein oractive fragment to mammalian cells, the method comprising incubating amammalian cell or membrane preparation with labeled polypeptide in thepresence of the drug and measuring the ability of the drag to block thisinteraction.

The use of a polynucleotide of the invention in genetic immunisationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356: 152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretrovital vectors (Seeger et al, PNAS 1984:81,5849). Suitable promotersfor muscle transfection include CMV, RSV, SRa, actin, MCK, alpha globin,adenovirus and dihydrofolate reductase.

In therapy or as a prophylactic, the active agent may be administered toa patient as an injectable composition, for example as a sterile aqueousdispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to human patients, it is expected that the dailydosage level of the active agent will be from 0.01 to 10 mg/kg,typically around 1 mg/kg. The physician in any event will determine theactual dosage which will be most suitable for an individual patient andwill vary with the age, weight and response of the particular patient.The above dosages are exemplary of the average case. There can, ofcourse, be individual instances where higher or lower dosage ranges aremerited, and such are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of a patientand remain in position for an extended time. Such devices include, forexample, artificial joints, heart valves, pacemakers, vascular grafts,vascular catheters, cerebrospinal fluid shunts, urinary catheters,continuous ambulatory peritoneal dialysis (CAPD) catheters, etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent staphylococcal wound infections.

Many orthopaedic surgeons consider that patients with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteraemia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5 ug/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks.

With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable patients.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing the cell surface protein.

In order to facilitate understanding of the following example certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

EXAMPLE 1 Isolation of DNA Coding for Novel Cell Surface Protein from S.Aureus WCUH 29

The polynucleotide having the DNA sequence given in SEQ ID NO 2 wasobtained from the sequencing of a library of clones of chromosomal DNAof S. aureus WCUH 29 in E. coli. In some cases the sequencing data fromtwo or more clones containing overlapping S. aureus WCUH 29 DNA was usedto construct the contiguous DNA sequence in SEQ ID No 2. Libraries maybe prepared by routine methods, for example:

Methods 1 and 2

Total cellular DNA is isolated from Staphylococcus aureus strain WCUH29(NCIMB 40771) according to standard procedures and size-fractionated byeither of two methods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolsed with a combination of fourrestriction enzymes (RsaI, PalI, AluI and Bsh1235I) andsize-fractionated according to standard procedures. EcoRI linkers areligated to the DNA and the fragments then ligated into the vector LambdaZapII that have been cut with EcoRI, the library packaged by standardprocedures, and E. coli infected with the packaged library. The libraryis amplified by standard procedures.

EXAMPLE 2 The Determination of Expression During Infection of a Genefrom Staphylococcus aureus WCUH29

Necrotic fatty tissue from a four day groin infection of Staphylococcusaureus WCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amouse animal model of infection

10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37 degrees C. for 16-20 hours. 4 week old mice (female, 18g-22 g, strain MF1) are each infected by subcutaneous injection of 0.5ml. of this broth culture of Staphylococcus aureus WCUH29 (diluted inbroth to approximately 10⁸ cfu/ml.) into the anterior, right lowerquadrant (groin area). Mice should be monitored regularly during thefirst 24 hours after infection, then daily until termination of study.Animals with signs of systemic infection, i.e. lethargy, ruffledappearance, isolation from group, should be monitored closely and ifsigns progress to moribundancy, the animal should be culled immediately.

Visible external signs of lesion development will be seen 24-48 h afterinfection. Examination of the abdomen of the animal will show the raisedoutline of the abscess beneath the skin. The localised lesion shouldremain in the right lower quadrant, but may occasionally spread to theleft lower quadrant, and superiorly to the thorax. On occasions, theabscess may rupture through the overlying skin layers. In such cases theaffected animal should be culled immediately and the tissues sampled ifpossible. Failure to cull the animal may result in the necrotic skintissue overlying the abscess being sloughed off, exposing the abdominalmuscle wall.

Approximately 96 h after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing/storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care should betaken not to penetrate the abdominal wall. Holding the skin flap withforceps, the skin is gently pulled way from the abdomen. The exposedabscess, which covers the peritoneal wall but generally does notpenetrate the muscle sheet completely, is excised, taking care not topuncture the viscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissuesamples

4-6 infected tissue samples (each approx 0.5-0.7 g) in 2 ml screw-captubes are removed from -80° C. storage into a dry ice ethanol bath In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue is treated for 100 seconds at 5000 rpm in order to achievebacterial lysis. In vivo grown bacteria require longer treatment than invitro grown S. aureus WCUH29 which are disrupted by a 30 secondbead-beat.

After bead-beating the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide.

200 microliters of chloroform is then added and the tubes shaken by handfor 15 seconds to ensure complete mixing. After 2-3 minutes at roomtemperature the tubes are spun down at 12,000×g, 4° C. for 15 minutesand RNA extraction is then continued according to the method given bythe manufacturers of TRIzol Reagent i.e.: The aqueous phase, approx 0.6ml, is transferred to a sterile eppendorf tube and 0.5 ml of isopropanolis added. After 10 minutes at room temperature the samples are spun at12,000×g, 4° C. for 10 minutes. The supernatant is removed and discardedthen the RNA pellet is washed with 1 ml 75% ethanol. A brief vortex isused to mix the sample before centrifuging at 7,500×g, 4° C. for 5minutes. The ethanol is removed and the RNA pellet dried under vacuumfor no more than 5 minutes. Samples are then resuspended by repeatedpipetting in 100 microliters of DEPC treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at -80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at -20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1×TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1×MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³² Plabelled oligonucletide probe specific to 16s rRNA of S. aureus (K.Greisen, M. Loeffelholz, A. Purohit and D. Leong. J.Clin. (1994)Microbiol. 32 335-351). An oligonucleotide of the sequence:

    5'-gctcctaaaaggttactccaccggc-3'

is used as a probe. The size of the hybridising band is compared to thatof control RNA isolated from in vitro grown S. aureus WCUH29 in theNorthern blot. Correct sized bacterial 16s rRNA bands can be detected intotal RNA samples which show extensive degradation of the mammalian RNAwhen visualised on TBE gels.

c) The removal of DNA from Staphylococcus aureus WCUH29 derived RNA

DNA was removed from 73 microliter samples of RNA by a 15 minutetreatment on ice with 3 units of DNAaseI, amplification grade (GibcoBRL, Life Technologies) in the buffer supplied with the addition of 200units of Rnasin (Promega) in a final volume of 90 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 microliters of DEPCtreated water with the addition of Rnasin as described in Method 1.

d) The preparation of cDNA from RNA samples derived from infected tissue

10 microliter samples of DNAase treated RNA are reverse transcribedusing a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 1 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/-RT samples are treated withRNaseH before proceeding to the PCR reaction

e) The use of PCR to determine the presence of a bacterial cDNA species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents:

45 microliters PCR SUPERMIX (Gibco BRL, Life Technologies).

1 microliter 50 mM MgCl₂, to adjust final concentration to 2.5 mM.

1 microliter PCR primers(optimally 18-25 basepairs in length and

designed to possess similar annealing temperatures), each primer at 10mM initial concentration.

2 microliters cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows:

5 minutes at 95° C., then 50 cycles of 30 seconds each at 94° C., 42° C.and 72° C. followed by 3 minutes at 72° C. and then a hold temperatureof 4° C. (the number of cycles is optimally 30-50 to determine theappearance or lack of a PCR product and optimally 8-30 cycles if anestimation of the starting quantity of cDNA from the RT reaction is tobe made).

10 microliter aliquots are then run out on 1% 1×TBE gels stained withethidium bromide with PCR product, if present, sizes estimated bycomparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies).Alternatively if the PCR products are conveniently labelled by the useof a labelled PCR primer (e.g. labelled at the 5'end with a dye) asuitable aliquot of the PCR product is run out on a polyacrylamidesequencing gel and its presence and quantity detected using a suitablegel scanning system (e.g. ABI Prism™377 Sequencer using GeneScan™software as supplied by Perkin Elmer)

RT/PCR controls may include +/-reverse transcriptase reactions, 16s rRNAprimers or DNA specific primer pairs designed to produce PCR productsfrom non-transcribed S. aureus WCUH29 genomic sequences.

To test the efficiency of the primer pairs they are used in DNA PCR withWCUH29 total DNA. PCR reactions are set up and run as described aboveusing approx. 1 microgram of DNA in place of the cDNA and 35 cycles ofPCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR:

1. Genes which are not transcribed in vivo reproducibly fail to give aproduct in RT/PCR.

2. Genes which are transcribed in vivo reproducibly give the correctsize product in RT/PCR and show a stronger signal in the +RT samplesthan the signal (if at all present) in -RT controls.

The following nucleotide sequence (SEQ ID NO 2) was identified in theabove test as transcribed in vivo. Deduced amino acid sequence is givenas SEQ ID NO 1. An example of a pair of PCR primers used to identify thegene are 5'-aattttatat gatatggc-3' SEQ ID NO 3! and 5'-aatgtatttgttaccttg-3' SEQ ID NO 4!.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetLysLysLeuValProLeuLeuLeuAlaLeuLeuLeuLeuValAla                              151015                                                                        AlaCysGlyThrGlyGlyLysGlnSerSerAspLysSerAsnGlyLys                              202530                                                                        LeuLysValValThrThrAsnSerIleLeuTyrAspMetAlaLysAsn                              354045                                                                        ValGlyGlyAspAsnValAspIleHisSerIleValProValGlyGln                              505560                                                                        AspProHisGluTyrGluValLysProLysAspIleLysLysSerThr                              65707580                                                                      AspAlaAspValIleLeuTyrAsnGlyLeuAsnLeuGluThrGlyAsn                              859095                                                                        GlyTrpPheGluLysAlaLeuGluGlnAlaGlyLysSerLeuLysAsp                              100105110                                                                     LysLysValIleAlaValSerLysAspValLysProIleTyrLeuAsn                              115120125                                                                     GlyGluGluGlyAsnLysAspLysGlnAspProHisAlaTrpLeuLys                              130135140                                                                     PheArgTyrGlyIleLysTyrValLysThrIleGlnGlnThrPheIle                              145150155160                                                                  AspThrThrLysAsnIleLysGlnIleMetGluLysGlnGlyAsnLys                              165170175                                                                     TyrIleAlaGlnLeuGluLysLeuAsnAsnAspSerLysAspLysPhe                              180185190                                                                     AsnAspIleProLysGluGlnArgAlaMetIleThrSerGluGlyAla                              195200205                                                                     PheLysTyrPheSerLysGlnTyrGlyIleThrProGlyTyrIleTrp                              210215220                                                                     GluIleAsnThrGluLysGlnGlyThrProGluGlnMetArgGlnAla                              225230235240                                                                  IleGluPheValLysLysHisLysLeuLysHisLeuLeuValGluThr                              245250255                                                                     SerValAspLysLysAlaMetGluSerLeuSerGluGluThrLysLys                              260265270                                                                     AspIlePheGlyGluValTyrThrAspSerIleGlyLysGluGlyThr                              275280285                                                                     LysGlyAspSerTyrTyrLysMetMetLysSerAsnIleGluThrVal                              290295300                                                                     HisGlySerMetLys                                                               305                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 930 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGAAAAAATTAGTACCTTTATTATTAGCCTTATTACTTCTAGTTGCTGCATGTGGTACT60                GGTGGTAAACAAAGCAGTGATAAGTCAAATGGCAAATTAAAAGTAGTAACGACGAATTCA120               ATTTTATATGATATGGCTAAAAATGTTGGTGGAGACAACGTCGATATTCATAGTATTGTA180               CCTGTTGGTCAAGATCCTCATGAATATGAAGTTAAACCTAAAGATATTAAAAAGTCAACT240               GACGCTGACGTTATTTTATACAACGGATTAAATTTAGAGACTGGTAACGGTTGGTTTGAA300               AAAGCCTTAGAACAGGCTGGTAAATCATTAAAAGATAAAAAAGTTATCGCAGTATCAAAA360               GATGTTAAACCTATCTATTTAAACGGTGAAGAAGGCAACAAAGATAAACAAGATCCACAC420               GCATGGTTAAAGTTTAGATATGGTATTAAATACGTAAAAACAATTCAACAAACATTTATC480               GATACGACAAAAAACATAAAGCAGATTATGGAAAAGCAAGGTAACAAATACATTGCTCAA540               TTGGAAAAATTAAATAATGACAGTAAAGACAAATTTAATGACATTCCAAAAGAACAACGT600               GCCATGATTACAAGTGAAGGTGCCTTCAAGTACTTCTCAAAACAATACGGTATTACACCA660               GGTTATATTTGGGAAATTAACACTGAAAAACAAGGTACACCTGAACAAATGAGACAAGCT720               ATTGAGTTTGTTAAAAAGCACAAATTAAAACACTTATTAGTAGAAACAAGTGTTGATAAG780               AAAGCAATGGAAAGTTTATCTGAAGAAACGAAGAAAGATATCTTTGGTGAAGTGTACACA840               GATTCAATCGGTAAAGAAGGCACTAAAGGTGACTCTTACTACAAAATGATGAAATCAAAT900               ATTGAAACTGTACACGGAAGCATGAAATAA930                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTTTATATGATATGGC18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AATGTATTTGTTACCTTG18                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide encoding a firstpolypeptide having at least 70% identity to a second polypeptide of SEQID NO: 1 wherein said first polypeptide is capable of generatingantibodies having binding specificity for said second polypeptide.
 2. Anisolated polynucleotide that is complementary over the entire length ofthe polynucleotide of claim
 1. 3. The polynucleotide of claim 1 whereinthe polynucleotide is DNA.
 4. The polynucleotide of claim 1 wherein thepolynucleotide is RNA.
 5. The polynucleotide of claim 3 comprisingnucleotides 1 to 930 set forth in SEQ ID:
 2. 6. The polynucleotide ofclaim 1 comprising the sequence set forth in SEQ ID NO: 2 that encodessaliva binding protein polypeptide.
 7. The polynucleotide of claim 1that encodes a polypeptide comprising amino acids 1 to 309 of SEQ IDNO:
 1. 8. An isolated polynucleotide encoding a first polypeptide havingat least 70% identity to a second polypeptide encoded by the DNAcontained in NCIMB Deposit No. 40771, said DNA having the polynucleotideof SEQ ID NO: 2 wherein said first polypeptide is capable of generatingantibodies having binding specificity for said second polypeptide.
 9. Anisolated polynucleotide that is complementary over the entire length ofthe polynucleotide of claim
 8. 10. A vector comprising the DNA of claim3.
 11. A host cell comprising the vector of claim
 10. 12. A process forproducing a polypeptide comprising: expressing from the host cell ofclaim 11 a polypeptide encoded by said DNA.
 13. A process for producinga cell that expresses a polypeptide comprising transforming ortransfecting the cell with the vector of claim 10 such that the cellexpresses the polypeptide encoded by the DNA contained in the vector.14. An isolated polynucleotide comprising a member selected from thegroup consisting of:(a) a polynucleotide having at least a 95% identityto a polynucleotide having SEQ ID NO: 2 encoding a polypeptidecomprising amino acids 1 to 309 of SEQ ID NO: 1; (b) a polynucleotidethat is complementary to the polynucleotide of (a).
 15. Thepolynucleotide of claim 14 wherein the polynucleotide is DNA.
 16. Thepolynucleotide of claim 14 wherein the polynucleotide is RNA.
 17. Thepolynucleotide of claim 15 comprising nucleotides 1 to 930 set forth inSEQ ID No:
 2. 18. The polynucleotide of claim 15 comprising the sequenceset forth in SEQ ID NO: 2 that encodes saliva binding proteinpolypeptide.
 19. The polynucleotide of claim 14 that encodes apolypeptide comprising amino acids 1 to 309 of SEQ ID NO:
 1. 20. Anisolated polynucleotide comprising a member selected from the groupconsisting of:(a) a polynucleotide having at least a 95% identity to apolynucleotide having SEQ ID NO: 2 encoding the same mature polypeptideexpressed by the DNA contained in NCIMB Deposit No. 40771 having thepolynucleotide sequence of SEQ ID NO: 2; (b) a polynucleotidecomplementary to the polynucleotide of (a).
 21. A vector comprising theDNA of claim
 15. 22. A host cell comprising the vector of claim
 21. 23.A process for producing a polypeptide comprising: expressing from thehost cell of claim 22 a polypeptide encoded by said DNA.
 24. A processfor producing a cell that expresses a polypeptide comprisingtransforming or transfecting the cell with the vector of claim 21 suchthat the cell expresses the polypeptide encoded by the DNA contained inthe vector.
 25. An isolated polynucleotide comprising a member selectedfrom the group consisting of:(a) a polynucleotide having at least a 97%identity to a polynucleotide having SEQ ID NO: 2 encoding a polypeptidecomprising amino acids 1 to 309 of SEQ ID 1; (b) a polynucleotide thatis complementary to the polynucleotide of (a).
 26. The polynucleotide ofclaim 25 wherein the polynucleotide is DNA.
 27. The polynucleotide ofclaim 25 wherein the polynucleotide is RNA.
 28. The polynucleotide ofclaim 26 comprising nucleotides 1 to 930 set forth in SEQ ID NO:
 2. 29.The polynucleotide of claim 26 comprising the polynucleotide sequenceset forth in SEQ ID NO: 2 that encodes saliva binding proteinpolypeptide.
 30. The polynucleotide of claim 25 that encodes apolypeptide comprising amino acids 1 to 309 of SEQ ID NO:
 1. 31. Anisolated polynucleotide comprising a member selected from the groupconsisting of:(a) a polynucleotide having at least a 97% identity to apolynucleotide having SEQ ID NO: 2 encoding the same mature polypeptideexpressed by the DNA contained in NCIMB Deposit No. 40771 having thepolynucleotide sequence of SEQ ID NO: 2; (b) a polynucleotidecomplementary to the polynucleotide of (a).
 32. A vector comprising theDNA of claim
 26. 33. A host cell comprising the vector of claim
 32. 34.A process for producing a polypeptide comprising: expressing from thehost cell of claim 33 a polypeptide encoded by said DNA.
 35. A processfor producing a cell that expresses a polypeptide comprisingtransforming or transfecting the cell with the vector of claim 32 suchthat the cell expresses the polypeptide encoded by the DNA contained inthe vector.
 36. An isolated polynucleotide comprising a polynucleotideencoding the polypeptide sequence of SEQ ID NO: 1.